Hi.
I have a protein quantification problem. I have several crude mitochondrial protein extracts, in which I have to quantify a particular protein of interest and show the extent of variation in quantity of the particular protein in each of the extract. Though the protein is an enzyme, for several reasons, I cannot do an enzyme assay for quantification. I have polyclonal antibodies against the protein of interest but I cannot do immunoprecipitation since I have very little of the antibody left and it is impossible to raise the polyclonal antibody again (that is a long story).
I have so far tried using western blots for quantification, but found it very unsatisfactory, since results are not very repetetive, and the slight variations in protein binding across the blot makes it very inconsistent and error prone. Moreover the protein of interest is very small (<20 kDa).
I would appreciate any idea or suggestion in this regards however absurd it may sound. As you can imagine, I am at my wit's end myself.
I was thinking of ELISA lately, but would use of polyclonal antibody be a good idea?
thank and I would appreciate any suggestion on this issue
Jai

I am stumped too! Here is an idea which may not be too outlandish. Your
experimental protocol permitting, you could label the protein in vivo (35S) for
your different mitochobdrial samples. Then do an autoradiography of the gel.
The autoradiogram can be used to show th quantitative variation across the gel
for your protein of interest (this is assuming that you can actually detect the
polypeptide on a gel. BTW, around 20kDa is not too small. I am not sure how
you could actually quantify any protein without some sort of stoichiometry.

Can you do ELISA with the antibody as a bait and then enzymatic assay of the
protein? That would work if the reason why you don't want to quantitate the
protein in the extract enzymatically is that there are contaminating
activities with your chosen assay reaction.
Otherwise, could you do at least 1-step fractionation of your extracts
(ammonium sulfate, organic extraction, chromatography...) and them
quantitate electrophoretically?
-Emir

""Jayakumar, R"" <[Only registered users see links. ]> wrote in message
news:97101976F8A044468CA74FE11883B90E0204898C@VIST A.roswellpark.org...
Hi.
I have a protein quantification problem. I have several crude
mitochondrial protein extracts, in which I have to quantify a particular
protein of interest and show the extent of variation in quantity of the
particular protein in each of the extract. Though the protein is an enzyme,
for several reasons, I cannot do an enzyme assay for quantification. I have
polyclonal antibodies against the protein of interest but I cannot do
immunoprecipitation since I have very little of the antibody left and it is
impossible to raise the polyclonal antibody again (that is a long story).
I have so far tried using western blots for quantification, but found
it very unsatisfactory, since results are not very repetetive, and the
slight variations in protein binding across the blot makes it very
inconsistent and error prone. Moreover the protein of interest is very
small (<20 kDa).
I would appreciate any idea or suggestion in this regards however
absurd it may sound. As you can imagine, I am at my wit's end myself.
I was thinking of ELISA lately, but would use of polyclonal antibody
be a good idea?
thank and I would appreciate any suggestion on this issue
Jai